Our objective is to develop innovative and sensitive immunoproteomic assays applicable to analysis of estrogen dependent proteins involved in reproductive processes. This technology employs an antibody capture step fottowed by identification of captured proteins by mass spectometry. Immnunoproteomics has the potential to allow rapid (<30 min) assays requiring small volumes of sample and antibody. Key to any immunoproteomic assay is optimizing the buffering systems for tissue extraction, the antibody capture step and washing of the immobilized antigen. This enhances the signal to noise ratio of the mass spectrometric ste and potentially obviate variations in protein-antibody interactions that will plague muItiple assay procedures. Towards this end we will initially develop the immunoproteomic technology for known estrogen markers, followed by measurement of those markers in the complex biological mixtures of cell extracts. Thus, we have two Specific Ams. 1. Develop an immunoproteomic assay for selected estrogen sensitive markers. 2. Demonstrate estrogenization with the technology in Specific Aim 1. Our studies will focus on the progesterone receptor and FKBP52. Cytosolic extracts from a variety of reproductive tissues including rabbit uterine cytosol, Ishakawa cells, and T47D breast cancer cells will be employed. Assorted buffers will be utilized to extract/solubilize proteins, optimize antibody capture and wash immobilized antgen prior to laser desorption. Mass spectral analyses of time-of-flight (TOF) utilized Ciphergen TM SELDI [surface enhanced laser desorption ionization] technology will detect the presence of the estrogen sensitive proteins. Applications include: assessment of action of environmental estrogens, screening for endocrine disruptors, a diagnostic tool for breast and other steroid sensitve carcinomas, study of anti-estrogens, steroid sensitivity profiling, and assessment of postranslatonal modifications.